In order to dissect the biochemical steps involved in genetic recombination we have chosen to focus on a key early step: strand exchange between homologous parental DNAs. In vitro, the product of this strand exchange reaction is a joint molecule composed a single- strand DNA joined to one end of a linear duplex DNA. We have established a new paradigm for this homologous pairing. In essence, that recombinases such as the E. coli RecA protein can form a novel DNA triplex (R-form DNA) in which the third strand may have any arbitrary sequence and must have a parallel orientation with respect to the phosphodiester backbone of the identical strand in the duplex. In order to understand the mechanism and structures involved in greater detail we have endeavored to miniaturize the reaction. In the past, we have shown that short oligonucleotides can be used as the substrates. Recently we have determined that a 20 amino acid peptide from RecA can promote the key reaction by the whole RecA protein: pairing (targeting) of a single stranded DNA to its homologous site on a duplex DNA. In the course of the reactions the peptide bound to both substrate DNAs, unstacked the single-stranded DNA, and assumed a b structure. Efforts are now underway to determine the three- dimensional structure of this peptide when bound to DNA. In order to improve the binding affinity of the peptide for DNAs and the efficiency of targeting by the peptide, we have mutagenized the corresponding 20 amino acid stretch in the whole RecA protein to determine those amino acid residues that can be mutated without loss of RecA function in vivo. We have shown that the synaptic complexes between three strands of DNA and RecA can be used to effect the selective cleavage of human DNA (RecA-Assisted-Restriction- Endonuclease (RARE) cleavage). Recently, we have able to demonstrate that such complexes can be used to direct sequence-specific ligation resulting in at least a 2000-fold enrichment of genomic DNA. We have designated this technique as RecA-Assisted-Cloning or RAC.